Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

Abstract

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5'-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.

Keywords: G protein-coupled receptor (GPCR); G protein-coupled receptor kinase; crystallography; phosphorylation; protein kinase; receptor regulation; signal transduction.

FIGURE 1.

Atomic structure of GRK5. A, thermostabilization of GRK5 induced by different ligands. Stability of GRK5 against thermal denaturation was monitored by circular dichroism measurement of changes in the ellipticity intensity at 223 nm as a function of temperature. The measurements were performed at 5 μm GRK5 in the absence (apo-GRK5) or presence of 0.4 mm ligand. B, GRK5·AMP-PNP·Mg²⁺ crystals were grown at 4 °C to dimensions 0.04 × 0.04 × 0.12 mm. The crystals show bipyramidal morphology in contrast to rod-like thin plate crystals of its close homolog GRK6 (28). C, domain organization and important functional regions of GRK5. The RH domain is shown in blue, the catalytic domain in yellow (N-lobe) and orange (C-lobe), and the C-tail of kinase domain in purple. The two clusters of positively charged residues at the N and C terminus encompass GRK5 CaM- and PIP₂-binding sites (magenta). N-terminal amphipathic α-helix is in green. Autophosphorylation sites on the C-tail of the kinase domain are indicated by yellow circles. D, ribbon representation of GRK5·AMP-PNP crystal structure. Full-length GRK5(1–590) was crystallized, and residues 15–543 are clearly resolved (the first and last residues are labeled). The color coding is the same as in C. AMP-PNP ligand and Mg²⁺ are shown as sticks and black sphere, respectively.

FIGURE 2.

RH domain displacement in GRK5. Comparison of the structural topology of the RH and kinase domains in GRK5·AMP-PNP and GRK6·sangivamycin (PDB code 3NYN). Kinase domains were superimposed to reveal an 8.5° rotation of RH domain. The comparison of GRK5 with the GRK1 and GRK2 crystal structures reveals similar displacements of structural elements within RH domain with largest changes in the RH bundle subdomain (6–13 Å of α6-helix displacement) (data not shown).

FIGURE 3.

GRK5 is a monomer both in the crystal structure and in solution. A, structural basis for monomeric state of GRK5. Atomic structures of GRK5·AMP-PNP (cyan) and GRK6·sangivamycin (chain A of dimeric complex PDB 3NYN is shown in pink) were aligned to highlight differences in the topology of the visible regions of their C termini. GRK5 is oriented from the top of the view in Fig. 1D, and its C terminus is shown in magenta, and the GRK6 C terminus is in red. The GRK6 C terminus is domain-swapped; its position is fixed by symmetry-related GRK6 molecule from the dimer complex (data not shown), and the GRK5 C terminus is aligned against its own RH domain. Key intramolecular contacts between the GRK5 C terminus and RH domain that support the specific architecture of the C terminus are highlighted in the left box, and the hydrophobic pocket consisting of Met¹⁶⁵, Phe¹⁶⁶, and Arg¹⁶⁹ (RH domain) that retains Phe⁵²⁷ (C terminus) is highlighted in the right box. Sequence alignment of C-terminal regions of human GRKs indicates unique Pro⁵²⁹ (framed in magenta) that forms a kink, thereby hindering a domain-swapped interface in GRK5. Identical residues are boxed in red, and residues showing similarity are in red and grouped in a blue frame. B, size-exclusion chromatographic analysis of purified GRK5. A Superose 12 16/60 gel filtration column was calibrated using molecular mass markers, whose elution volumes and relative molecular masses were used to build a calibration curve (inset). GRK5 eluted as a single species consistent with an ∼70-kDa monomeric state of the protein. C, sedimentation-velocity profile of GRK5. The top panel shows the raw absorbance (solid circles) collected at 276 nm with the interval of 40 min together with corresponding fitted data (solid line) plotted as a function of radial position. The low values (< 5% of signal) for residuals confirm fidelity of fit between raw and fitted data. The monophasic sedimentation boundaries suggest that GRK5 exists as a monodisperse single species in solution. The bottom panel shows the fitted distribution of the apparent sedimentation coefficient calculated for GRK5 is 2.6 S, which corresponds to an estimated molecular mass of ∼62.4 kDa, consistent with a monomer of 67.8 kDa (theoretical molecular mass).

FIGURE 4.

GRK5 kinase domain adopts a partially closed conformation. A, structural alignment of the GRK5 kinase domain with the open, partially closed (intermediate), and fully closed conformations of the kinase domains from PKA (top panel) and GRK6 (bottom panel) reveals that the GRK5 kinase domain is in an intermediate, partially closed conformation. Structural coordinates of the kinase domain C-lobe were superimposed to reveal conformational changes in the N-lobe. The changes in the N-lobe that are indicative of kinase domain closure (αB, αC, αK, and P-loop) are shown with red arrows. Only AMP-PNP (stick model) from GRK5, which indicates active site of kinase, is shown. Inset, GRK5 in complex with AMP-PNP or sangivamycin (active site view) was aligned based on their C-lobes to highlight 1 Å lowering of P-loop (slightly more closed conformation) in the sangivamycin complex compared with the AMP-PNP complex. B, ligand-binding pockets for AMP-PNP and sangivamycin in GRK5. Left panel, view of catalytic cleft with residues that form ligand-binding site presented in stick model. The one magnesium ion (black sphere) is retained in tridentate manner by oxygen atoms of α-, β-, and γ-phosphates of AMP-PNP with the side chains of Asp³²⁹ (DLG motif) and Asn³¹⁶ also involved in its coordination. The catalytic H₂O molecule that mediates Asn³¹⁶ and Asp³²⁹ contact with Mg²⁺ and the triphosphate tail is shown as a red sphere. Nitrogen, oxygen and phosphorus atoms are colored blue, red, and orange, respectively. Right panel, two-dimensional schematic diagrams of GRK5 interactions with AMP-PNP and sangivamycin. Residues that form hydrogen bonds (dotted lines) with the ligands are shown in ball-and-stick with the interatomic distances shown in Å. The spoked arcs represent residues making van der Waals interactions with the ligand. Diagrams were generated with Ligplot plus. C, differences in conformation of triphosphate tail of AMP-PNP in crystal structures of GRK5 and GRK6 (PDB code 2ACX). The conserved residue Glu²³⁴ (αC-helix) hydrogen bonds with another conserved residue, Lys²¹⁵ (β3-strand), in GRK5 and GRK6 (shown as cyan dashed line between GRK5 residues). Lys²¹⁵ pairs with β-phosphate of AMP-PNP in GRK6 (yellow dashed lines), although β-phosphate in GRK5 is positioned far from Lys²¹⁵. D, P-loop/αB-helix binding interface in GRK5·AMP-PNP structure. Main chain oxygen of Gly¹⁹⁶ (P-loop) is engaged in hydrogen-bonding network, including Arg²²⁵, Arg²²¹, and Glu²¹⁸ of αB-helix (N-lobe). Residues involved in the binding interface are shown in ball-and-stick representation, and AMP-PNP and Mg²⁺ are shown as stick and black sphere, respectively.

FIGURE 5.

AST is an integral part of the nucleotide-binding pocket of GRK5. A, sequence alignment of human GRK kinase domain C-terminal extension. GRK5 autophosphorylation sites (Ser⁴⁸⁴ and Thr⁴⁸⁵) are indicated as is Arg⁴⁷⁰ in the AST (framed in magenta), which forms a direct contact with the nucleotide in GRK5. Identical residues are boxed in red, and residues showing similarity are in red and grouped in a blue frame. CLT, C-lobe tether. B, interaction of AST with nucleotide in the active site of GRK5 and PKA as viewed from above the P-loop. Phe³²⁷ and Tyr³³⁰ (AST) mediate van der Waals interactions with the AMP-PNP purine and 2′-OH of the ribose ring (via active-site conserved water), respectively, in PKA, and the guanidinium group of Arg⁴⁷⁰ hydrogen bonds with the 3′-OH of AMP-PNP ribose ring in GRK5. The β1–β3 strands were removed for clarity. C, hydrogen bond network stabilized by Arg⁴⁷⁰ in GRK5 complexes with AMP-PNP and sangivamycin. Arg⁴⁷⁰ mediates interactions with the nucleotide (via ribose ring of AMP-PNP and sangivamycin), N-lobe (via Arg¹⁹⁰ and Leu¹⁹²), hinge (via Asn²⁶⁷), and C-lobe (via Asp²⁷⁰ in GRK5·sangivamycin). Thus, Arg⁴⁷⁰ likely plays an important role in the interface that promotes closure of the kinase domain and regulates nucleotide entry/exit. The GRK5·AMP-PNP structure lacks direct Asp²⁷⁰ (C-lobe) contact with Arg⁴⁷⁰ and ribose (mediated by catalytic water molecule in this case) that is observed in GRK5·sangivamycin and might explain the slightly less closed conformation of the kinase domain in GRK5·AMP-PNP. D, interactions within the catalytic site of PKA bound to AMP-PNP and substrate peptide (magenta) analogous to Arg⁴⁷⁰ in GRK5. Side chain of Arg⁴⁷⁰ is modeled in yellow ball-and-stick representation to show its position in GRK5 relative to PKA (kinase domains of GRK5 and PKA are aligned). Active site conserved water molecule (red sphere) that connects Tyr³³⁰ to the ribose ring of AMP-PNP in PKA hydrogen bonds with Leu⁴⁹ (β1-strand of N-lobe) and the ribose ring hydroxyl group, thus resembling the guanidinium group of Arg⁴⁷⁰ interactions with Leu¹⁹² and 3′-OH of the AMP-PNP ribose in GRK5. Moreover, the AST/hinge and the AST/N-lobe contacts provided by backbone carbonyl and amide of Arg⁴⁷⁰ in GRK5 are similar to Asn³²⁶ (AST)/Ala¹²⁴ (hinge) and Asp³²⁹ (AST)/Lys⁴⁹ (N-lobe) contacts, respectively, in PKA. Thus, interactions of AST with the nucleotide and N- and C-lobes that help to stabilize the closed state of the kinase domain in PKA are arranged in a similar manner in GRK5 and PKA. E, effect of R470A mutation on Michaelis-Menten kinetics for ATP. The kinetic parameters were determined by incubating 50 nm GRK5, 10 μm rhodopsin, and 2–200 μm ATP as described under “Experimental Procedures.” The data are derived from three experiments and fitted to Michaelis-Menten kinetics using GraphPad Prism.

FIGURE 6.

Structural mobility of the kinase domain C-tail in GRKs. A, comparison of kinase domain C-tail orientation in GRK5 relative to its position in GRK6 (PDB code 3NYN, upper left), GRK1 (PDB code 3C4W, lower left), and PKA (PDB code 3TNP, lower right) reveals a large displacement of the GRK5 NLT segment compared with GRK1 and GRK6. The GRK5 NLT moves away from the catalytic cleft and occupies a position close to the RH domain with autophosphorylation sites (red sticks) largely shifted from their position in GRK1 and GRK6 (23 Å and 26 Å, respectively). The structural coordinates of the GRK5 NLT more closely resemble the conformation of the NLT in PKA (12 Å distance between corresponding positions of autophosphorylation sites, Ser³³⁸ in PKA and Ser⁴⁸⁴ in GRK5). Inset, upper right, Tyr⁴⁷³, which is conserved in the GRK1 and GRK4 subfamilies, forms a hydrogen bond with the backbone amide of Asp⁴⁶⁸ and stabilizes the tail loop (AST loop) in GRK6. In GRK5, Tyr⁴⁷³ shifts 12 Å away from its position in GRK6 and does not stabilize formation of the AST loop. B, GRK5 is autophosphorylated in an intra- as well as intermolecular manner. GRK5 (WT) and kinase-dead GRK5-K215R (KD) were autophosphorylated alone or in a WT/KD mixture at a 1:5 molar ratio. The reaction was done in the presence of [γ-³²P]ATP and phospholipid vesicles at 30 °C for 2 or 60 min as described under “Experimental Procedures.” Only intramolecular autophosphorylation of WT GRK5 was observed after 2 min, which demonstrates the mobility of the GRK5 kinase domain C-tail, which can access the active site of GRK5 situated ∼25 Å away from the catalytic cleft in the crystal structure. Phosphorylation of the KD mutant was only detected at 60 min when WT GRK5 was present, demonstrating the ability of GRK5 to autophosphorylate in an intermolecular manner. GRK5 that is fully autophosphorylated (F), intermediately autophosphorylated (I), or nonphosphorylated (N) is denoted on the right.

FIGURE 7.

GRK5 electrostatic surface and a model for GRK5 binding to phospholipid membranes. A, electrostatic surface potential of GRK5. The surface potential of GRK5 is depicted from −5 (red, acidic) to +5 (blue, basic) kT/e⁻, and neutral regions are white. The stretch of basic residues is clustered on the top of GRK5 and maps the presumed membrane-binding surface. The N-terminal membrane-binding region (residues 22–35) is abundant in Lys residues (labeled) and is at the core of the interface providing electrostatic interaction of GRK5 with negatively charged lipids in the plasma membrane. A few positively charged residues of the N-lobe and the C-tail of kinase domain might also contribute to electrostatic anchoring of GRK5 on membranes. It should also be emphasized that although the C-terminal membrane-binding region (residues 546–562) is not resolved in the GRK5 crystal structure, it is near the last residue that was observed (Pro⁵⁴³, labeled) and thus should be in the same plane with the N-terminal membrane-binding region likely constituting a common electrostatic surface for membrane attachment. The electrostatic contour of the kinase-active site also has a basic character. Substrate peptide (yellow stick model) from a PKA·AMP-PNP·SP20 complex (PDB code 4DG0) was mapped onto GRK5 C-lobe to model substrate binding into the catalytic cleft of GRK5 (coordinates of C-lobes of GRK5 and PKA were aligned). The zoomed-in view demonstrates the peptide orientation relative to AMP-PNP in GRK5. The site of phosphorylation on the substrate peptide is labeled and is positioned against the γ-phosphate of AMP-PNP. This illustrates the initial step of the kinase catalytic cycle (substrate and nucleotide alignment into the kinase-active site). B, structural model depicting binding and CaM-driven dissociation of GRK5 from phospholipid membrane and GPCRs. The model is based on the GRK5 crystal structure, which is oriented relative to the phospholipid surface in a manner that would bring the N-terminal membrane domain (residues 22–35) in close contact with PIP₂ in the plasma membrane. Color coding is the same as in Fig. 1D. The GRK5 C terminus (residues 544–590) is incomplete in the crystal structure and therefore unresolved regions are depicted as a gray dashed line, and the C-terminal membrane-binding region (residues 546–562) is depicted as a gray α-helix. The crystal structure of GRK5 suggests close location of both N- and C-terminal membrane-binding determinants implying that they can form a single interface and work synergistically to target GRK5 to the membrane. Binding of agonist to inactive GPCR (light green, from the crystal structure of β₂AR in complex with the inverse agonist ICI 118551, PDB code 3NY8) promotes GPCR activation. Docking of GRK5 on an activated GPCR (green, from the β₂AR·G_s complex structure, PDB code 3SN6) promotes full closure of the kinase domain and proper alignment of phosphoacceptor sites on the receptor (green dashed line). CaM and GRK5 autophosphorylation regulate the stability of GRK5 association to the membrane and GPCR. CaM (shown as a brown model) drives GRK5 redistribution from membrane compartment to cytoplasm or nucleus, where GRK5 targets a different set of substrates. GRK5 autophosphorylation sites (Ser⁴⁸⁴ and Thr⁴⁸⁵) are located at the presumed GRK5/lipid bilayer interface in the crystal structure, and hence, autophosphorylation might change the electrostatic potential of GRK5 and destabilize GRK5 anchoring on the membrane.